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  • Supplementary Materials for The influence of maternal high fat diet during lactation on offspring hematopoietic priming

    work
    Creator:
    Singer, Kanakadurga
    Description:
    Supplementary Figure 1. Example gating scheme for bone marrow mature leukocytes and hematopoietic progenitors. To stain for mature leukocytes antibodies used were against CD45, Ly6G, CD11b, CD115, CD19, and CD3e. All CD45+ cells were gated first. Neutrophils were defined as Ly6G+CD11b+, monocytes were defined as Ly6G-CD11b+CD115+ (17,18), B cells were defined as Ly6G-CD11b-CD19+, and T cells were defined as Ly6G-CD11b-CD3e+. To stain for hematopoietic stem and progenitor cells antibodies used were against lineage panel (B220, Gr1, TER119, CD11b, CD4, CD8), cKit, Sca1, CD48, CD150, CD16/32, and CD105. HSCs were defined as Lin-Sca1+cKit+CD48-CD150+, MPPs were defined as Lin-Sca1+cKit+CD48-CD150-, HPC1 were defined as Lin-Sca1+cKit+CD48+CD150-, HPC2 were defined as Lin-Sca1+cKit+CD48+CD150+, GMP were defined as Lin-Sca1-cKit+CD150-CD16/32+, PreGM were defined as Lin-Sca1+cKit+CD150-CD105-, preMegE were defined as Lin-Sca1+cKit+CD150+CD105-, and PreCFUe were defined as Lin-Sca1+cKit+CD150+CD105+. , Supplementary Figure 2. Hematopoietic stem and progenitor cell frequency by flow cytometry as a percentage of CD45 bone marrow cells in male and female Ctrl and HFD PN offspring. Hematopoietic stem cells (HSC), multipotent progenitor cells (MPP), Pre-Granulocyte Macrophage (Pre-GM), granulocyte monocyte precursors (GMP), Pre-Megakaryocyte-Erythroid Precursors (Pre-MegE) and erythroid precursors (Pre-CFUE). Analyses were Student’s t-test ang gating per Supplementary Figure 1., Supplementary Table 1. Differential gene expression between Ctrl and HFD PN male gonadal white adipose tissue (GWAT) from postnatal day 16. The significant gene expression differences were determined by the DESeq2 package for R Studio. Sequencing The RNA was extracted from adipose tissue using Trizol LS (Life Technologies) by Qiagen RNeasy Mini Kit (74106) according to the manual. The RNA was sent to the University of Michigan Advanced Genomics Core for RNA-sequencing. For RNAseq studies, gonadal white adipose tissue 3’ QuantSeq single-end poly-A mRNA libraries were generated (Lexogen). These were sequenced to a depth of 10-20M reads on Illumina NovaSeq platform. Data are available from GEO at accession number GSE227337., and Supplementary Table 2. Differentially expressed genes between Ctrl and HFD PN male gonadal white adipose tissue (GWAT) from postnatal day 16 that are significant after correction for false discovery rate were determined by the DESeq2 package for R Studio.
    Citation to related publication:
    Kim K, Varghese M, Sun H, Abrishami S, Bowers E, Bridges D, Meijer JL, Singer K* and Gregg B*. The influence of maternal high fat diet during lactation on offspring hematopoietic priming. Endocrinology.  https://doi.org/10.1210/endocr/bqad182 PMID 38048597.
    Discipline:
    Health Sciences

  • Supplementary Materials for Sex differences in inflammatory responses to adipose tissue lipolysis in diet-induced obesity

    work
    Creator:
    Singer, Kanakadurga
    Description:
    Supplementary Figure 1. Tissue weights in response to HFD feeding and CL treatment. (A) GWAT weight as % of body weight (B) IWAT weight as % of body weight (C) BAT weight as % of body weight (D) Liver weight as % of body weight. N=7-12 /group; *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001; error bars are SEM. Comparisons of M ND PBS vs F ND PBS are shown as #p<0.05, ##p<0.01, ###p<0.005 and M HFD PBS vs F HFD PBS are shown as &p<0.05, &&p<0.01, &&&p<0.005, &&&&p<0.0001., Supplementary Figure 2. Free glycerol estimation in lean and obese male and female WAT and BAT depot explants with ADRB3 stimulated lipolysis. Free glycerol estimation in lean and obese (A) GWAT (B) IWAT (C) BAT explant tissues. Free glycerol released calculated as fold change over basal conditions in lean and obese (D) GWAT (E) IWAT (F) BAT explant tissues. N=8 /group; *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001; error bars are SEM. Comparisons of M ND PBS vs F ND PBS are shown as #p<0.05, ##p<0.01, ###p<0.005 and M HFD PBS vs F HFD PBS are shown as &p<0.05, &&p<0.01, &&&p<0.005, &&&&p<0.0001., Supplementary Figure 3. Gene expression (A) Akt1 and (B) Glut4 gene expression in obese male and female GWAT with and without ADRB3 stimulation. A.U., arbitrary units, N=5-8; *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001., Supplementary Figure 4. Flow cytometry assessment of ATMs in lean and obese IWAT SVF. Quantitation of (A) IWAT percent ATMs (B) IWAT CD11c+ ATMs (C) IWAT CD11c-ATMs (D) IWAT dendritic cells (DC) numbers, N=7-12/group; *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001. , Supplementary Figure 5. Lipidomic assessment of lipid mediators in obese male and female GWAT. (A) Relative hydroxy fatty acids (FAHFA) (B) Phosphatidylserine (PS) (C) Phosphatidylcholine (PC) (D) Lyso-PC (E) Phosphatidylethanolamine (PE) (F) Phosphatidylglycerol (PG) (G) Phosphatidylinositol (PI) content in obese male and female GWAT with and without CL treatment. N=6/group; *p<0.05, **p<0.01, ***p<0.005, ****p<0.0001., and Curation note: Title of deposit changed from "Supplementary Materials Lipolysis Paper" Dec. 20, 2018 to more closely reflect association with the paper these materials support.
    Discipline:
    Science